Stem cell biology will play a significant role in the repair and regeneration of tissues and organs damaged by disease or injury. Therefore a better understanding for the molecular events that serve to regulate maintenance of stem cell progenitor populations, as well as identifying the signaling pathways that determine stem cells into lineage specific differentiation will remain important issues in biomedical research. Neural crest cells are multipotent stem cells that contribute to a diverse array of tissues throughout the embryo. Determination of the fate of mammalian neural crest has been hindered by the lack of an in vitro culture system. Amelogenin besides being a structural protein indispensable for enamel formation appears to also function as a signaling molecule in cell specification. Amelogenin splicing products have been shown to exert specific signaling effects and to induce changes in cell differentiation both in vitro and in vivo. We propose that amelogenin is a signaling molecule capable of modulating embryonic stem cell differentiation to neural crest cell fate and subsequently along osteogenic lineage. Using embryonic stem cells, we will test this hypothesis by executing the following specific aims: I) Using loss-of-function and gain-of-function experiments to determine the biological function of amelogenin in directing embryonic stem (ES) cell differentiation to cranial neural crest (CNC)-like multipotent progenitor cells; II) Using a tissue recombination strategy to examine the potential of cranial neural crest like multipotent progenitor cells derived from ES cells to differentiate to odontogenic lineage. The successful derivation of cranial neural crestlike multipotent progenitor cells from embryonic stem cells will provide a new tool for cell lineage analysis of neural crest in vitro. The results from these specific aims will be used to optimize strategies for maintenance of stem cell populations while improving our ability to stimulate the development of cell specific lineages needed for the repair and regeneration of tissue and organ defects in craniofacial and skeletal tissues. [unreadable] [unreadable] [unreadable]